Oxidant-Induced S-Glutathiolation Inactivates Protein Kinase C-α (PKC-α):  A Potential Mechanism of PKC Isozyme Regulation

Protein kinase C (PKC) isozymes are subject to inactivation by reactive oxygen species (ROS) through as yet undefined oxidative modifications of the isozyme structure. We previously reported that Cys-containing, Arg-rich peptide−substrate analogues spontaneously form disulfide-linked complexes with PKC isozymes, resulting in isozyme inactivation. This suggested that PKC might be inactivated by oxidant-induced S-glutathiolation, i.e., disulfide linkage of the endogenous molecule glutathione (GSH) to PKC. Protein S-glutathiolation is a reversible oxidative modification that has profound effects on the activity of certain enzymes and binding proteins. To directly examine whether PKC could be inactivated by S-glutathiolation, we used the thiol-specific oxidant diamide because its oxidant activity is restricted to induction of disulfide bridge formation. Diamide weakly inactivated purified recombinant cPKC-α, and this was markedly potentiated to nearly full inactivation by 100 μM GSH, which by itself was without effect on cPKC-α activity. Diamide inactivation of cPKC-α and its potentiation by GSH were both fully reversed by DTT. Likewise, GSH markedly potentiated diamide inactivation of a PKC isozyme mixture purified from rat brain (α, β, γ, ε, ζ) in a DTT-reversible manner. GSH potentiation of diamide-induced cPKC-α inactivation was associated with S-glutathiolation of the isozyme. cPKC-α S-glutathiolation was demonstrated by the DTT-reversible incorporation of [35S]GSH into the isozyme structure and by an associated change in the migration position of cPKC-α in nonreducing SDS−PAGE. Diamide treatment of NIH3T3 cells likewise induced potent, DTT-reversible inactivation of cPKC-α in association with [35S] S-thiolation of the isozyme. Taken together, the results indicate that PKC isozymes can be oxidatively inactivated by S-thiolation reactions involving endogenous thiols such as GSH.